Chemically modified silicas have been, and continue to be, widely used as supports in a great variety of chromatographic separations. With the aim of controlling its selectivity while reducing unwanted interactions with one or more compounds, numerous synthetic procedures have been developed to attach organic moieties (R) on the silica surface. Early work on the chemical modification of silica (Halasz and Sebastian, Angew. Chem. (Int. Ed.) 8:453 (1969); Deuel et al., Helv. Chim. Acta 119:1160 (1959)) described the use of an esterification reaction between surface silanol groups (SiOH) and an alcohol to give a structure of the following type: ##STR1## Although such materials were useful for many separations, their limited hydrolytic stability seriously precluded the extensive usage of these bonded phases, particularly in liquid chromatography which requires the use of aqueous eluents.
Currently, commercially available bonded phases are prepared by reacting selected organosilanes with the silica surface. Halogen- or alkoxy-substituted alkyldimethylsilanes are the most commonly used silanizing reagents. The resulting bonded support bears monolayer surface structures of the following type: ##STR2## By changing the structure of the R group, it is possible to produce bonded silicas with a great variety of organic groups, ranging from non-polar materials, for instance, octyl- and octadecyl-silicas commonly used as bonded supports in reversed-phase liquid chromatography, to ionic materials such as benzenesulphonic acid derivatives which are widely used in ion-exchange liquid chromatography. The preparation of these and similar materials are described in a number of publications (e.g., Roumeliotis and Unger, J. Chromatogr. 149:211 (1978) or Asmus et al. J. Chromatogr. 123:109 (1976)) and patents (Sebastian et al. U.S. Pat. No. 3,956,179; Hancok et al. U.S. Pat. No. 4,257,916; or Ramsden et al. U.S. Pat. No. 4661,248).
In a related approach, polymeric (multilayer) bonded stationary phases are prepared from bi- or tri-substituted organosilanes with the general formula X.sub.n SiR.sub.4-n, where X=alkoxy, halide or any easily hydrolyzed group, and n=2,3. The resulting polymeric bonded support bears repeating surface structures of the type ##STR3## where Y=--R (n=2) or --O-- (n=3) and the oxygen atom (--O--) is bonded either to a hydrogen (that is, as part of a free silanol, Si--O--H) or to another silicon atom (that is, as part of a siloxane linkage, Si--O--Si). A number of patents and publications describe the preparation of these materials (Kirkland and Yates, U.S. Pat. Nos. 3,722,181 (1973), and 3,795,313 (1974); Novotny et al., J. Chromatog. 83:25 (1973); Sander and Wise, Anal. Chem. 56:504 (1984)). Although in many instances these bonded supports provide satisfactory separations, the lack of control of the polymerization process seems to be a major contributor to such problems as irreproducible layer thickness and incomplete silanol condensation. This limitation has confined polymeric bonded stationary phases to applications where the presence of a multilayer is necessary and/or its thickness is relatively unimportant. As a consequence, the vast majority of liquid chromatographic separations are carried out with monolayer bonded phases.
The recent development of electrophoretic separations in a capillary format has promoted the extent of the silanization technology normally used in chromatography to the deactivation of the inner wall of the fused silica capillary. Thus, Jorgenson et al. (Science 222:266 (1983)) have noted that separation of model proteins, such as cytochrome, lysozyme and ribonuclease A, in untreated fused silica capillaries with a phosphate buffer at pH 7 was accompanied by severe tailing, and suggested that this might be caused by strong interactions between the proteins and the capillary wall. Derivatization of the capillary wall has been proven effective to prevent or control protein adsorption (McCormick, Anal. Chem. 60:2322 (1988); Bruin et al., J. Chromatog. 471:429 (1989)). In addition, by chemically modifying the inner surface of the capillary, operational variables such as the electrosomotic flow are more amenable to control. In another application (Hjerten, U.S. Pat. No. 4,680,201 (1987); Cohen and Karger, U.S. Pat. Nos. 4,865,706 and 4,865,707 (1989)), a method is described for preparing fused-silica capillary tubes for electrophoretic separations by use of a bifunctional compound in which one group (usually a terminal --SiX.sub.3 group where X=ethoxy, methoxy or chloride) reacts with the capillary wall and the other (usually an olefin group) does so with a monomer taking part in a polymerization process. This process resulted in a wall-bonded, polymer-filled capillary useful for polyacrylamide gel electrophoresis.
The extensive usage of these bonded materials in chromatography and capillary electrophoresis does not necessarily imply that they meet all requirements with respect to separation performance and stability. On the contrary, monomeric bonded phases, for instance, are subject to serious effects arising primarily from a relatively limited organic coverage due to the "bulky" methyl groups of the anchored moiety, and from a still unsatisfactory hydrolytic stability of the Si--O--Si--C linkage, particularly under moderately acidic or slightly alkaline elution conditions. Similarly, polymeric bonded phases although having somewhat better organic coverages, contain a considerable population of free silanols and also exhibit a limited hydrolytic stability. Incomplete surface coverage and poor hydrolytic stability both result in the exposure of a substantial number of surface silanols, groups which are known to be primarily responsible for the residual adsorption phenomena that plague silicon-based separation materials. These so called "silanophilic" interactions are usually undesirable in chromatography as well as in capillary electrophoresis because they often result in "tailing" peaks, catalyze solute decomposition, lead to unreliable quantitation, etc. One of the most striking cases of silanophilic interactions occurs perhaps in the separation of certain compounds containing amino or other similar groups, particularly biomolecules. For instance, many proteins may interact very strongly with unreacted silanols leading to excessive band tailing, incomplete recovery of one or more solutes, or even recovery of the same component from different bands.
In an effort to overcome such problems, other organosilane reagents have been developed. Two related approaches have been proposed in which either the methyl groups of the organosilane reagent are replaced by bulkier groups (Glajch and Kirkland, U.S. Pat. No. 4,705,725, (1987)) or a "bidentate" silanizing reagent is used (Glajch and Kirkland, U.S. Pat. No. 4,746,572, (1988)). In both cases the new groups are aimed to shield the unreacted silanols as well as the hydrolytically labile linkage that bonds the silane to the support. Although this steric protection has resulted in somewhat improved bonded phases, the synthetic procedures still involve the formation of unstable Si--O--Si--C linkage, and therefore, the necessity still exists for a truly effective silane chemistry.
In another completely different approach, bonded silicas bearing direct Si--C linkages have been developed. They involve the sequential reaction of the silica substrate with a chlorinating reagent (e.g., thionyl chloride) and a proper alkylating reagent (e.g., a Grignard or organolithium compound): ##STR4## where --M=--Li or --MgBr. In principle, this method should provide not only a closer attachment and a denser coverage of organic functionalities but also a more hydrolytically stable bonded phase than that obtained by the corresponding Si--O--Si--C linkage. However, the acceptance for the application of a chlorination/Grignard or chlorination/organo-lithium reaction sequence as a routine method to modify silica substrates has been hindered by several factors. One factor is that the one-step organosilanization procedure (such as described in U.S. Pat. No. 3,956,179 to Sebastian et al.) is relatively easy to carry out as compared to the two-step halogenation/alkylation sequence. Difficulties associated with the removal of residual salts which may be occluded in the porous silica matrix during the alkylation process is also an important factor which has contributed to the limited usage of this synthetic approach. Finally, but not less importantly, the preparation of the alkylation reagent exhibits strong interferences with many reactive functionalities, particularly those containing carbonyl, nitrile, carboxyl, amide, alcohol, etc. That is, the great reactivity which makes a Grignard reagent so useful in many synthetic approaches seriously limits its applicability. The organic group, R, in the Grignard reagent, RMgBr, must remain intact during the preparation of the reagent. It is a well known fact that Grignard reagents react with acidic components to form the corresponding hydrocarbon group R--H. More strictly, "any compound containing hydrogen attached to an electronegative element such as oxygen, nitrogen, and even triply-bonded carbon are acidic enough to decompose a Grignard reagent" (Morrison and Boyd, Organic Chemistry, 3rd Edition, 1974). Additionally, a Grignard reagent reacts readily with molecular oxygen, carbon dioxide, and with "nearly every organic compound containing a carbon-oxygen or carbon-nitrogen multiple bond" (supra). The nitro group (--NO.sub.2) also appears to react oxidatively with a Grignard reagent. It seems clear therefore that only a very limited number of organic functionalities may be present in the halide compound from which a Grignard reagent can be prepared. Being even more reactive than the corresponding Grignard reagent, an organolithium reagent should exhibit the same limitations described above to a similar or even greater extent. This, of course, greatly limits the versatility of this approach.
It is therefore desirable to address the shortcomings of existing bonded packings by developing an alternate silane chemistry which combines the superior coverage and hydrolytic stability of direct Si--C linkages with the preparation simplicity of silanization.